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genome editing reference guide

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Perform gene knock-in by homology-directed repairWith the Alt-R™ CRISPR-Cas system

OVERVIEW

The quickest way to make a precise genome modification is by using a Cas enzyme, a guide RNA (gRNA), and a donor DNA template.

The following list provides the main requirements for CRISPR gene knock-in HDR experiments (see also Figure 1):

1. Cas enzyme

2. Guide RNA

3. HDR donor template

4. Cells

5. Delivery method (choose 1)

a. Electroporation

b. Lipofection

c. Microinjection

6. Positive control guide RNA

7. (Optional) Alt-R Genome Editing Detection Kit

Analyze your editing results

Select or designa guide RNA

and donor template

Select a Cas enzyme Form an RNP complex and prepare

donor template

Deliver genomeediting reagents

Figure 1. CRISPR HDR workflow.

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Perform gene knock-in by homology-directed repair 2


PROTOCOL GUIDE

Select a Cas enzyme

The CRISPR-Cas system has 2 components:

1. Cas enzyme—an RNA-guided endonuclease (e.g., Cas9 or Cas12a)

2. Guide RNA

Cas enzymes require an RNA molecule, known as the guide RNA, to guide the enzyme to a specific location in the genome, which is referred to as the target DNA. The guide RNA contains an approximately 20 nucleotide sequence called the spacer that is complementary to the target DNA protospacer. Downstream to the complementary sequence in the target DNA is the protospacer-adjacent motif (PAM), which is recognized by the Cas enzyme. Both a guide RNA and a PAM are required for the Cas enzyme to bind to the target DNA and subsequently create a double- strand break (DSB).

Whether you use Cas9 or Cas12a will depend on the PAM sites that are available in the target region of the genome you want to study. Cas9 recognizes an NGG PAM, while Cas12a recognizes TTTV (V = A/C/G). Cas9 is better suited for GC rich regions of the genome, while Cas12a is better for AT-rich regions. See Table 1 for a direct comparison between the 2 enzymes to help you decide which to use in your application.

When using Cas9

For Cas9, you can pick between IDT’s Alt-R S.p. Cas9 Nuclease V3 or Alt-R S.p. HiFi Cas9 Nuclease V3 as the enzyme for targeting genomic regions with NGG sequences. For most experiments, the Alt-R S.p. Cas9 Nuclease V3 sufficiently provides efficient genome editing. If you are concerned about off-target effects, use the HiFi Cas9 enzyme for the most precise editing.

IDT also offers 2 Cas9 nickases: Alt-R S.p. Cas9 D10A Nickase V3 creates a single-strand cut in the targeted strand of DNA, and Alt-R S.p. Cas9 H840A Nickase V3 creates a single-strand cut in the non-targeted strand of DNA. To use in genome editing experiments, a single nickase is used with 2 guide RNAs to create a double-strand break. To learn how to use nickases, review this DECODED article.

When using Cas12a

Considering Cas12, you can pick between IDT’s Alt-R A.s. Cas12a (Cpf1) V3 or Alt-R (Cpf1) Cas12a Ultra as the enzyme for targeting genomic regions with TTTV sequences. Cas12a (Cpf1) Ultra enzyme is the result of protein engineering and directed evolution. With these enhancements, this enzyme is as reliable as the Cas9 nuclease.

https://www.idtdna.com/pages/education/decoded/article/when-and-how-to-use-nickases-for-efficient-genome-editing



Table 1. Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1).

Cas9 system Cas12a system

tracrRNA

Cas9 crRNA

5’

PAM

3’

Cas12a (Cpf1)

PAM

crRNA

5’3’

Applications General genome editing• General genome editing when additional

flexibility is needed and Cas9 is not a suitable choice.

• For species with AT-rich genomes

Ribonucleoprotein components

• gRNA options: 1. crRNA and tracrRNA 2. sgRNA

• Cas9 endonuclease

• crRNA• Cas12a endonuclease

Alt-R CRISPR enzymes• Wild-type• HiFi (reduces off-targeting editing) [1]• D10A Nickase

• Wild-type• Ultra (improves performance)

Cas9 crRNA:tracrRNA

crRNA• Native: 42 nt• Alt-R: 35–36 nt

tracrRNA• Native: 89 nt• Alt-R: 67 nt

—

Cas9 sgRNA • Alt-R: 99–100 nt —

Cas12a crRNA — • Native: 42–44 nt• Alt-R: 40–44 nt

CRISPR enzyme• Class 2, Cas type II• M.W.*: 162,200 g/mol• Endonuclease domains: RuvC-like and

HNH

• Class 2, Cas type V• M.W.*: 156,400 g/mol• Endonuclease domain: RuvC-like only

Double-stranded DNA cleavage

• Wild-type and HiFi: blunt-ended cut 3 bases upstream of the protospacer sequence

• D10A nickase with paired crRNAs: 5’ overhang

• H840A nickase with paired crRNAs: 3’ overhang

• PAM site often destroyed during genome editing

• 5’ overhanging cut on the 5’ side of the protospacer sequence

• PAM site may be preserved after genome editing

PAM sequence† NGG • TTTV for Cas12a V3 and Cas12a UltraCurrent recommendations for Alt-R RNP delivery

• Electroporation with (optional) Alt-R Cas9 Electroporation Enhancer

• Microinjection• Lipid-mediated transfection

• Electroporation with Alt-R Cas12a Electroporation Enhancer (recommended)

• Microinjection

* Molecular weight of Alt-R nuclease

† N = any base; V = A, C, or G



Select or design a guide RNA

The guide RNA will direct the Cas enzymes to the target site where the enzyme generates a DSB. The break is repaired by a cellular DNA repair mechanism that follows 1 of 2 main pathways:

1. Non-homologous end joining (NHEJ), where broken ends of the DNA are efficiently joined, but often with insertions or deletions at the breakpoint, which mainly leads to gene knock-out (on rare occasions, indels could occur in-frame leading to the production of proteins with partial function)

2. Homology-directed repair (HDR), where cells use a DNA template, provided along with CRISPR components, to repair the DNA break via homologous recombination

IDT provides predesigned guide RNAs for Cas9 for the genomes of 5 species: human, mouse, rat, zebrafish, and C. elegans with guaranteed performance.

Note: If a predesigned guide RNA for the genome you want to look at is not available, you can use IDT’s design tool to custom design your own guide.

The guide RNA can be ordered as 2 separate parts (crRNA and tracrRNA) and combined before the experiment, or as a single guide RNA (sgRNA) that has the 2 parts already combined. All types of Alt-R RNAs are chemically modified to prevent immune stimulation and nuclease degradation.

2-part guide RNA consists of crRNA and tracrRNA. crRNA (dark blue in Figure 2A) provides the sequence specificity through an approximately 20 nt region, known as the spacer, that is complementary to the target DNA. The remaining sequence of the crRNA (16 nt) is complementary to tracrRNA (green in Figure 2A). The tracrRNA is a universal RNA molecule that does not change for each new target sequence but must be bound to the crRNA to create a functional, targeting ribonucleoprotein (RNP) with the enzyme.

A. Alt-R crRNA:tracrRNA B. Alt-R sgRNA

tracrRNA

Cas9 crRNA

5’

PAM

3’

67 nt universal tracrRNA36 nt site specific crRNA

Cas9crRNA

5’

PAM

3’

linker loop

tracrRNA

100 nt site specific sgRNA

Figure 2. Comparison between 2-part guide RNA delivery system and a 1-part guide.

Alternatively, you can use an sgRNA, a 1-part fusion of the crRNA and tracrRNA molecules (Figure 2B).

https://www.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM



When deciding between the 2-part versus the sgRNA, there are several differences to consider. The Alt-R 2-part guide RNA is composed of 2 shorter RNA molecules, a 35–36 nucleotide crRNA and a 67 nucleotide universal tracrRNA. Both molecules are chemically modified to prevent immune stimulation and nuclease degradation.

Tip: Shorter RNA molecules cut costs because you only need to order this 35–36 nucleotide site-specific crRNA for every new sequence that you would like to target.

For sgRNA, you have a 100-nucleotide, site-specific sgRNA for every sequence that you want to target. These are chemically modified guide RNAs to prevent immune stimulation and nuclease degradation.

With both 2-part and single guide RNA, similarly high editing levels are achieved. For a smaller number of target sites, one or the other type of guide RNA may provide better editing efficiency.

Note: If you are using Cas12a as your enzyme, you will need to use only a crRNA. With Cas12a, you should design 2–3 target sequences as described in our CRISPR genome editing DECODED article.

When selecting a gRNA for HDR experiments there are several considerations. It is imperative that the selected guide has high editing efficiency to enable high frequency of HDR. In addition, you may want to select a guide with low potential off-target cleavage to avoid undesired off-target effects. The choice of which nuclease to use should be dependent on where the relative genomic locations of the desired mutation reside in relation to the available CRISPR-Cas guides. For Cas9 nuclease, the guide should cut as close as possible to the desired HDR mutation. For Cas12a, the optimal insertion occurs at positions 12–16 of the guide sequence. When using the Cas9 D10A nickase, the HDR mutation should be placed between the two nick sites.

Design a donor template

IDT offers 2 formats for ssDNA donor templates:

1. Alt-R Donor Oligos—chemically modified single-stranded DNA up to 200 bases

2. Megamer ssDNA fragments—single-stranded DNA from 201 to 2000 bases long

To enable homology-based recombination, the HDR repair mechanism requires that the donor DNA contains regions of homology to both sides of the double-strand break. This donor DNA harboring overlaps of sufficient lengths must be delivered simultaneously with the Cas ribonucleoprotein (RNP) complex (formed by the Cas endonuclease and the targeting RNA system). We recommend 30–60 nt lengths for homology arms for short single-stranded oligo deoxynucleotide (ssODN) donors (e.g., Alt-R Donor Oligos;



The addition of phosphorothioate bonds (PS) on each end of a ssODN template increases HDR efficiency over unmodified ssODN templates. These modifications (see Figure 3) are beneficial in some circumstances. However, we have also developed Alt-R HDR Donor Oligos which include 2 PS linkages and an IDT proprietary end-blocking modification at each end to provide increased stability. Our data show that use of the Alt-R HDR modification results in the highest HDR efficiency.

Alt-R HDRmodified

PSmodified

Unmodified NNNNNNNNNNNN NNNNNNNNNNNN

NNNNNNNNN NNNNNNNNNPS PS PSPS N N NNAlt-RHDRAlt-RHDR

NNNNNNNNN NNNNNNNNNPS PS PSPS N N NN

Figure 3. Modifications for the highest HDR efficiency.

The introduction of silent blocking mutations into the protospacer sequence or mutating the PAM site itself when an intact CRISPR recognition site is present on the HDR template will help prevent the HDR template from reconstituting the gRNA recognition site, and avoid unwanted re-cutting at the same locus after the repair. The Alt-R HDR Design Tool allows for silent mutation incorporation using empirically defined rules to provide the highest HDR rates.

Deliver genome editing reagents

Once you have chosen your CRISPR reagents, you must choose a method of delivery. Transfer efficiency and subsequent cell viability are essential considerations when making your choice between methods.

For the most efficient genome editing, we recommend using an RNP consisting of Cas9 or Cas12a nuclease in complex with guide RNA (crRNA:tracrRNA duplex or sgRNA, or crRNA respectively). Using this combination provides very high editing efficiency across most target sites and addresses issues (e.g., inconsistent Cas enzyme expression levels and incorporation of DNA expression constructs) that can be problematic with other CRISPR-Cas editing methods. For more information about using RNP for CRISPR editing, see this protocol for Cas9 and this protocol for Cas12a.

With the RNP complex formed, deliver the complex to the cells with 1 of the following methods:

• Electroporation

• Lipofection

• Microinjection

Electroporation

Electroporation is the most commonly used method and the one we recommend for most experiments performing a standard CRISPR workflow. IDT also provides an enhancer to increase electroporation editing efficiency. Alt-R Electroporation Enhancer is specifically designed either for Cas9 or Cas12a (Cpf1) and acts as a carrier to transport the RNP complex more efficiently into the cells.

Include Alt-R Electroporation Enhancer (Cas9 or Cas12a) with the RNP complex and run electroporation. See this protocol for a detailed description of the steps involved for Cas9 RNP delivery with a donor template.

Important! Electroporation Enhancer can result in increased toxicity when used with Megamer ssDNA donor template, especially at high doses and longer Megamer fragments.

https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/alt-r-crispr-cas9-user-guide-ribonucleoprotein-transfections-recommended.pdf?sfvrsn=1c43407_24]https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cpf1-user-guide-rnp-electroporation-amaxa-nucleofector-system.pdf?sfvrsn=ec43407_14https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/homology-directed-repair-using-the-alt-r-crispr-cas9-system-and-hdr-donor-oligos.pdf?sfvrsn=47121607_6



Lipofection

Although electroporation is the preferred delivery method, lipofection can be used for delivery, especially if you already have a high-efficiency lipofection protocol for delivering molecules into cells. Lipofection is primarily suitable for CRISPR experiments in easy-to-transfect cells, such as some adherent, immortalized eukaryotic cell lines. Refer to this protocol for more details on using lipofection for delivery.

Microinjection

Microinjection is another method of delivering CRISPR components. Usually this method is used for delivery to embryos. In comparison to electroporation and lipofection, it is a more labor-intensive, time-consuming, and costly method that requires highly skilled lab personnel and specialized equipment.

Alt-R HDR Enhancer

For HDR experiments, we recommend using the Alt-R HDR Enhancer, which is a small molecule compound that has demonstrated an ability to increase the rate of HDR. While the efficiency of HDR and relative improvement in HDR rates varies by cell line, editing site, and the desired insert, we have guidelines that maximize HDR potential, and limit cytotoxicity often associated with the delivery of HDR Enhancer and genome editing reagents into cells.

Guidelines when using HDR Enhancer

• Use a maximum of 1% by volume DMSO in the final media.

• Use a control sample with DMSO, but no HDR Enhancer, in the final media to monitor toxicity.

• Use a concentration of 20–30 µM of HDR Enhancer in the final media.

• Change to growth media without HDR Enhancer 12–24 hours after electroporation.

Important! The optimal concentration for Alt-R HDR Enhancer will be cell type dependent and may require a dose titration. Toxicity should be monitored closely when used at concentrations higher than 30 µM.

Analyze your editing results

All 3 workflow options result in the delivery of RNP complex and HDR donor template into cells. Upon entry, your guide RNA leads the Cas enzyme to the target site where the enzyme cuts the cellular DNA. Once the double-strand break is introduced by the CRISPR system, the genomic DNA repair mechanism follows the NHEJ or HDR pathway. At this point, the genomic DNA is isolated from the cells to verify the editing events.

Measuring success with the CRISPR process

Did CRISPR successfully knock out the gene? In some cases, you may know in advance that an obvious phenotype will occur in the cells when you knock out a gene, and that may be all you need to confirm that CRISPR worked.

Though in most cases, sequencing is the best way to look specifically for mutations.

Positive controls

To ensure your experimental success, IDT has validated controls that consist of both a guide RNA (available as the two-part crRNA:tracrRNA or the single guide sgRNA) and a single-stranded HDR donor template, which are intended to serve as positive controls in your HDR experiments. See this document for more information about using CRISPR-Cas9 HDR positive controls.

https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/alt-r-crispr-cas9-user-guide-ribonucleoprotein-transfections-recommended.pdf?sfvrsn=1c43407_24https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/supplementary-product-info/idt_using-crispr-cas9-hdr-positive-controls.pdf?sfvrsn=b0371507_4



How can IDT help?

IDT offers reagents for both traditional Sanger sequencing and for next-generation sequencing (NGS). We also offer the T7EI assay (Alt-R Genome Editing Detection Kit), which can quickly confirm mutagenesis has occurred, but this assay is not as sensitive or specific as NGS. After you have nominated the likely hotspot sites of off-target effects, you can then verify off-target editing events using the IDT rhAmpSeq amplicon sequencing system (a proprietary targeted sequencing method of hotspot regions).

Workflow summary

Cas9: 2-part guide RNA workflow

1. Prepare a 2-part gRNA complex by annealing the 2 oligos:

a. Dilute the crRNA and tracrRNA to the desired concentrations in Nuclease-Free IDTE.

b. Prepare the gRNA by combining crRNA and tracrRNA at equimolar ratio to the desired concentration, heating the mixture 95°C for 5 minutes, then cooling it to room temperature (15–25°C) on the benchtop.

2. Dilute the donor template to the desired concentrations in Nuclease-Free IDTE.

3. Prepare the RNP complex by mixing gRNA with Alt-R S.p. Cas9 or HiFi Cas9 Nuclease V3, then incubate at room temperature for 10–20 minutes.

4. Transfect cells of interest by electroporation, or your delivery method of choice (Figure 4).

15 minutes

Step 1—Anneal to form gRNA

10–20 minutes

Step 2—Complex gRNA and Cas9 to form RNP

30–60 minutes

Step 3—Deliver RNP and donor template

Add donor template

Figure 4. RNP delivery with Cas9 nuclease and 2-part guide RNA.

Cas9: Single guide RNA workflow

1. Dilute the sgRNA to the desired concentration in Nuclease-Free IDTE.


3. Prepare the RNP complex by mixing sgRNA with Alt-R S.p. Cas9 or HiFi Cas9 Nuclease V3, then incubate at room temperature for 10–20 minutes.


10–20 minutes

Step 1—Complex gRNA and Cas9 to form RNP

30–60 minutes


Add donor template

Figure 5. RNP delivery with Cas9 nuclease and sgRNA.

https://www.idtdna.com/pages/products/crispr-genome-editing/alt-r-genome-editing-detection-kithttps://www.idtdna.com/pages/products/next-generation-sequencing/amplicon-sequencing



Cas12a workflow

1. Resuspend Alt-R CRISPR-Cpf1 crRNA to the desired concentration in Nuclease-Free IDTE.


3. Prepare the RNP complex by mixing gRNA with Alt-R S.p. Cas9 or HiFi Cas9 Nuclease V3, then incubate at room temperature for 10–20 minutes.


10–20 minutes

Step 1—Complex cRNA and Cas12a to form RNP

Add donor template

+crRNACas12a (Cpf1)

30–60 minutes


Figure 6. RNP delivery with Cas12a nuclease and crRNA.

REFERENCES

1. Vakulskas C, Dever D, et al. (2018) A novel high-fidelity Cas9 delivered as a ribonucleoprotein complex enables high frequency gene editing in human haematopoietic stem and progenitor cells. Nat Med 24(8):1216–1224. DOI 10.1038/s41591-018-0137-0.

Additional resources

See these additional resources for more information on performing and refining your CRISPR experiments:

• Protocol: Alt-R CRISPR-Cas9 System-RNP transfections

• Protocol: Alt-R CRISPR-Cas9 System-RNP electroporation, Nucleofector system

• Protocol: Alt-R CRISPR-Cas9 System-RNP electroporation, Neon Transfection system

• Protocol: Alt-R CRISPR-Cpf1-RNP electroporation, Nucleofector system

• Protocol: Alt-R CRISPR-Cpf1-RNP electroporation, Neon Transfection system

• Protocol: Homology-directed repair using the Alt-R CRISPR-Cas9 System and Megamer ssDNA Fragments

• Protocol: Homology-directed repair using the Alt-R CRISPR-Cas9 System and HDR Donor Oligos

• DECODED article: Do you have the best guide RNA (gRNA) for your CRISPR-Cas9 genome editing?

• Analysis guidelines: Evaluate CRISPR DNA editing with rhAmpSeq sequencing data

• IDT’s FAQ support web page on CRISPR genome editing

This information and more is available at www.idtdna.com/CRISPR.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107069/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107069/https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/alt-r-crispr-cas9-user-guide-ribonucleoprotein-transfections-recommended.pdf?sfvrsn=1c43407_24https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cas9-user-guide-ribonucleoprotein-electroporation-amaxa-nucleofector-system6a01611532796e2eaa53ff00001c1b3c.pdf?sfvrsn=71c43407_30https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cas9-user-guide-ribonucleoprotein-electroporation-neon-transfection-system0601611532796e2eaa53ff00001c1b3c.pdf?sfvrsn=6c43407_28https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cpf1-user-guide-rnp-electroporation-amaxa-nucleofector-system.pdf?sfvrsn=ec43407_14http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cpf1-user-guide-rnp-electroporation-neon-system.pdf?sfvrsn=9c43407_16https://www.idtdna.com/pages/education/decoded/article/do-you-have-the-best-guide-rna-(grna)-for-your-crispr-cas9-genome-editinghttps://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/analysis-guideline_evaluate-crispr-dna-editing-with-rhampseq-sequencing-data.pdf?sfvrsn=faf30507_23https://www.idtdna.com/pages/support/faqs?categories=crispr-genome-editinghttps://www.idtdna.com/CRISPR

Perform gene knock-in by homology-directed repair

Technical support: [email protected]

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> SEE WHAT MORE WE CAN DO FOR YOU AT WWW.IDTDNA.COM.

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